System for electrochemical quantitative analysis of analytes within a solid phase

ABSTRACT

The analytical process utilized in the system of this invention comprises three (3) step distinct steps wherein an analyte of interest in a test sample is initially labeled and subsequently isolated within a porous medium of a test device. Once isolated within the medium, the label is displaced from the analyte, or from the complex with the analyte, and converted, under electrolytic condition, to a metallic species which is caused test to deposit upon a working electrode of the test device. This working electrode is part of an electrode array that is positioned coincident with the porous medium, yet maintained physically remote therefrom. This deposit is then stripped from the working electrode, under anodic stripping conditions, and the current generated within the electrode array monitored. The characteristic response curve that is produced thereby can be correlated with the identity and concentration of the analyte(s) with the test sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of application U.S. Ser. No.09/305,771, filed May 5, 1999, now U.S. Pat. No. 6,485,983, issued Nov.26, 2002, which is a U.S. National Stage Entry under 35 USC § 371 ofinternational application PCT/US00/12099, filed May 5, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a system, test method and test devicewhich combines affinity-chromatographic test technique andelectrochemical quantitative technique for analysis of one or moreanalytes in a test sample. More specifically, this invention is directedto an electrochemical quantitative analysis system for determination ofan analyte (e.g. proteins, hormones or enzymes, small molecules,polysaccharides, antibodies, nucleic acids, drugs, toxins, viruses orvirus particles, portions of a cell wall and other compounds which havespecific or characteristic markers that permit their identification)within a solid phase (e.g. test strip) by measurement of electrochemicalchanges of label(s) associated with a labeled substance that can becorrelated with the concentration of the analyte of interest. The teststrip used in this analytical system includes a solid phase affinitychromatographic medium, having an electrochemical detection systemassociated therewith, within the fluid pathway through said medium. Theinventions hereinafter set forth in detail are applicable toquantitative analysis of virtually any analyte, whose concentration canbe correlated with an electroactive species of an electroactivesubstance that is capable of manifesting a change in electrical responsewithin the electrochemical cell-like environment of the test device ofthis invention.

2. Description of Prior Art

A. Immunodiagnostics—Immunochemical analysis for an analyte within asolid phase (test strip) generally involves an assay in which ananalyte, in a liquid sample, and a labeled substance, interact with eachother, or another reaction constituent. Within the context of the priorart, it is understood that a “labeled substance” can, thus, include (a)an analyte mimic that is labeled with an indicator, (b) a labeled ligandthat is specific for interaction with the analyte of interest, or (c) acomplex that is formed by interaction of (a) or (b) and yet anotherreaction constituent. The liquid containing the labeled substance andthe analyte is then transferred to a porous film or membrane, where itis drawn by diffusion or capillary action along a fluid pathway of themembrane, and encounters one or more companion test kit reagents (atleast one of which is immobilized within a definitive area thereof).This labeled substance and/or the analyte is thereafter captured by animmobilized companion test kit reagent at a test site to form a complex.If the complex is present in sufficient concentration, it produces adiscernible change at such site. Where the indicator is a pigment, suchas a metal (e.g. colloidal gold), or alternatively, a colored latexparticle, the discernible change is generally visible to the naked eye.

The following patents are regarded as representative of this type ofimmunochromatographic assay. These patents are arranged in chronologicalorder and no significance is to be attached to their order of discussionas to the patentability of the invention described and claimed herein.

Campbell et al, U.S. Pat. No. 4,703,017 (assigned to Becton Dickinson)describes a test method for detection of an analyte with a “direct” or“particulate” indicator, specifically an indicator that can be visuallydetected with the naked eye, thus, making such test method acceptablefor home diagnostic use; or, alternatively, in a clinical setting devoidof sophisticated analytical equipment. The direct indicator of choice isa liposome containing a pigment or equivalent colorant. The claimed testmethod is described as suitable for urine analysis of analytes (e.g.pregnancy test); and, thus, capable of detection of an elevated level ofanalyte. Accordingly, such assay is regarded as a semi-quantitativedetermination of the analyte of interest. Because the test involves asingle step (e.g. application of the sample to the test strip), the testhas become characterized in the art as a “Rapid” test.

Rosenstein, U.S. Pat. No. 5,591,645 (assigned to Becton Dickinson)describes an immunochromatographic test method based upon the principlesdescribed in commonly assigned U.S. Pat. No. 4,703,017 (to Campbell etal). The Rosenstein test device incorporates all of the test kit reagentin the “same plane” of the test strip and contemplates applying thesample directly to the test strip to reconstitute the reagents containedtherein and, thereby effect the desired analysis. The direct indicatorof choice can be selected from a number of equivalent colorants,however, colloidal gold adsorbed to a binding protein (e.g. antibody orantigen) is generally preferred. The claimed test method is described assuitable for urine analysis of analytes (e.g. pregnancy test); and,thus, capable of detection of an elevated level of analyte or asemi-quantitative determination of the analyte of interest. Because thetest also involves a single step (e.g. application of the sample to thetest strip), the test has become characterized in the art as a “Rapid”test.

Swanson et al, U.S. Pat. No. 5,073,484 (assigned to Abbott laboratories)describes an immunochromatographic test method utilizing multiple testzones, with each zone arranged along a common linear fluid pathway. Asthe test sample and test kit reagents migrate along this fluid pathway,the analyte of interest (generally associated with a direct indicator)becomes bound to an immobilized companion reagent in each of the testzones. Each of the test zones is precalibrated in reference to astandard, and thus the appearance of color in specific test zonecorrelated with the concentration level of analyte in the sample.Accordingly, the concentration of the analyte in the sample can beestimated from the level of color development along the fluid pathway ofthe test device. Because the test also involves a single step (e.g.application of the sample to the test strip), the test has becomecharacterized in the art as a “Rapid” test.

May et al, U.S. Pat. No. 5,602,040 (assigned to Unilever PatentHoldings) describes an immunochromatographic test method analogous tothe test methods described in the Campbell et al and Rosenstein patentsdiscussed above. One of principles differences identified and claimed byMay et al, is the lyophilization of a sugar, along with the directindicator, in the sample receiving site. The sugar addition isreportedly required to effect reconstitution of the lyophilizedindicator and thus permit its interaction with the analyte in thesample.

With the limited exception of the adaptation of the foregoing RapidTests to detection of an elevated level of analyte, these Rapid Testsare generally unsuitable for precise quantitative monitoring an analytelevel, or for the quantitative detection and differentiation of morethan one analyte within a single test environment.

B. Instrumental Analysis—Analysis of fluid samples has alsotraditionally involved the use of instrumentation and the measurement ofchanges in electrical potential and/or current at given site/electrode.Typically, the electrical measurement is referenced to a secondelectrode, and a number of measurements made over a given intervaland/or defined range of conditions.

The following patents are regarded as representative of this type ofelectrochemical analysis. These patents are arranged in chronologicalorder and no significance is to attach to the order of discussion, or totheir relative significance to the patentability of the inventiondescribed and claimed herein.

Wang U.S. Pat. No. 5,292,423 (assigned to New Mexico State UniversityTechnology Transfer Corp.) describes a method and apparatus for tracemetal analysis of fluid samples (e.g. drinking water, blood, urine etc.)by means of initial adsorption of the trace metal from a fluid sampleonto a screen printed carbon (working) electrode that had beenpre-coated with mercury. The Wang test platform also employs at leastone additional (reference) electrode. The electrode having the tracemetal was, thereafter, subjected to analysis by either Anodic StrippingVoltammetry (ASV) or Potentiometric Stripping Analysis (PSA). Thereported advantages of the Wang method and apparatus include the abilityto adapt his invention to on-site field testing of sample fluids forsuspected pollutants with a disposable testing device.

Brooks U.S. Pat. No. 5,753,517 (assigned to the University of BritishColumbia) describes a quantitative immunochromatographic assay utilizingan apparatus for detection of a particulate latex indicator inaccordance with the procedures associated with so called RAMP™technology. According to Brooks et al., the RAMP™ technology utilizeslatex particles in a manner similar to enzymes in ELISA assays. In theRAMP™ technology based analysis, a test strip, having two distinctfluorescent labeled indicators, is placed in a cartridge, and a fluidsample applied to the test strip. The analyte of interest interacts withone of the indicators forming a fluorescent complex which is captured inthe test zone of the test strip, and while the second indicator remainsin tact within the test strip, (so as to provide an internal standard).The RAMP™ technology is, thus, capable of differentiation of thefluorescent complex from the internal standard, and the amount ofanalyte in the sample determined thereby.

Brooks et al. alternative system for performance of his analysis,contemplate the use of optical detection methods (light scattering) andmeasurement of changes in electrical conductivity or resistively. In oneof the suggested alternatives, Brooks et al. contemplates (U.S. Pat. No.5,753,517-col. 7, lines 7-19, inclusive) the quantitative measurement ofthe analyte of interest by the electrochemical detection of a releasedelectroactive agents, such as bismuth, gallium or tellurium ions, from acomplex associated with the analyte of interest. According to Brooks etal one of these alternatives involves the use of a chelatingagent-protein conjugate as an indicator for the analyte of interest, anddetection thereof contemplates the addition of an acidic solution forrelease the metal label as ions for later quantitation by anodicstripping voltammetry (as described by Hayes et al, Anal. Chem.66:1860-1865 (1994).

The following published technical literature is regarded asrepresentative of this type of electrochemical analysis. These papersare arranged in chronological order and no significance is to attach totheir order of discussion, or to their relative significance to thepatentability of the invention described and claimed herein.

Electrochemical techniques have provided challenges associated withaffinity-chromatographic reactions, because of their stable andsensitive signal, lower level of detection limit, simple operation andcost-effectiveness. Pioneering studies by Henieman et al.(Hayes, F. H.et al., Anal. Chem., 66, 1860-1865 (1994)) illustrated the use of metalion labels for heterogeneous immunoassays with Anodic StrippingVoltammetric (ASV) detection. Such immunoassays involved covalentlylinking chelating agent to a protein to serve as a chelon for the metallabel. Following competitive equilibrium between the labeled andunlabeled protein for the antibody (immobilized on the surface of apolyester tube), the metal label was released and transferred to anelectrochemical cell for an ASV detection with a hanging mercury dropelectrode (HMDE) and a deaerated solution. Similarly, Wang et al. (WangJ., Anal. Chem., 70, 1682-1685 (1998)) employed an antibody-coated,screen-printed sensor, performed the entire assay protocol directly onthe surface of the disposable strip, and employed the highly sensitivepotentiometric stripping mode for detecting the released metal ion labelin microliter solutions. As desired for decentralized sensingapplications, such an on-chip protocol offers several advantagescompared to conventional ASV-based immunoassays, including simplifiedoperation (e.g., the elimination of the separation and reagent volumes,elimination of toxic mercury drops, and a more sensitive strippingdetection mode). The Wang et al. system requires relatively longincubation periods for pre-conditioning the sensor (prior to use) andfor interaction with the sample; and, multiple washing steps, both ofwhich make this method impractical and cumbersome.

OBJECTS OF THE INVENTION

It is the object of this invention to remedy the above and relateddeficiencies in the prior art.

More specifically, it is the principle object of this invention to adaptrapid affinity chromatographic electrochemical analysis to quantitativedetermination of analytes of interest.

It is another object of this invention to provide a system forelectrochemical quantitative analysis of an analyte(s) within a solidphase test format that is comparable in ease of use to rapidimmuchromatographic assays.

It is yet another object of this invention to provide a system forelectrochemical quantitative analysis of an analyte(s) within a solidphase test format involving a simplified means for detection of labelsby potentiostatic or potentiometric measurement of changes within thesolid phase that are attributable to the concentration(s) of label(s)within the test site.

It is still yet another object of this invention to provide a system forconcurrent electrochemical quantitative analysis of a common test samplefor multiple analytes within a solid phase test format involving asimplified means for detection of label(s) by stripping voltammetry

SUMMARY OF THE INVENTION

The above and related objects are achieved with the electrochemicalquantitative analysis system, method and test strip of this inventionfor determination of the concentration of an analyte within a solidphase test environment. Initially, a test sample solution and a labeledsubstance are initially contacted, under assay conditions, within asolid phase test environment, and caused to migrate along a fluidpathway therein. Irrespective of the assay format (competitive assay,sandwich assay, etc.), the labeled substance is concentrated (bound)within a delimited area of the solid phase. Within the context of thesystem and analytical method of this invention, a “labeled substance”can include any suitable electroactive material that can be isolatedwithin a delimited area of the solid phase, under assay conditions, orwhich can release an electroactive component within such delimited area,(also collectively hereinafter referred to as the “electroactivespecies”); and, such electroactive species thereafter, undergo anelectrochemical transition (e.g. redox) incidental to electrochemicalquantitative analysis. The observed measurement (transition) of theelectroactive species can be directly or inversely related, (e.g. bycomparison to a standard curve), to the concentration of the analyte ofinterest in the test sample.

The two principal types of electroanalytical measurements fordetermination of the concentration of the analyte of interest arepotentiometric and potentiostatic. In each of these analyticalprotocols, two electrodes are required/involved, and a contacting sample(electrolyte) solution, which together constitute an electrochemicalcell-like environment within the test strip. The electrode surface is,thus, the junction between the ionic conductor (e.g. electrolytes fromthe aqueous fluid sample, test kit reagents, etc.) and an electronicconductor. Within this electrochemical cell-like environment of the teststrip, the electrode that responds to the analyte of interest (or anelectroactive substance that is indicative of the analyte of interest),is termed the “indicator” or “working” electrode, whereas the electrodethat is maintained at a constant potential is termed the “reference”electrode (its response being independent of the sample solution).

In one of the embodiments of this invention, the immobilized labeledsubstance is contacted with a release reagent to initially cause therelease/displacement of the label from the immobilized labeled substancewithin this delimited area. In the case of a metal label, for example,the released/displaced metal can be further interacted with thesubstance in the release reagent solution, to form an metal film (ormetal surface-active complex) on the surface of the working electrodewithin the delimited area of the solid phase. Thereafter, the delimitedarea of the affinity chromatographic test strip is subjected topotentiostatic measurement, by anodic stripping voltammetry of the labelfrom the metal film (or metal surface-active complex) on the workingelectrode, which causes the label, to undergo yet a secondelectrochemical transition, (conversion of the label from the reducedform to the oxidized state). This second electrochemical transition ofthe label (from the reduced to the oxidized state) has a characteristicfingerprint that can be monitored and which, when compared to a standardcurve, can correlate directly with the concentration of the analyte inthe sample.

The stripped label from the metal film (or metal surface-active complex)and, thus, the analyte of interest, can be quantified by measurement ofchanges within the delimited area of the test strip by potentiostaticelectrochemical techniques. The specifics of this electrochemicalquantitative analysis, involve the application of an electricalpotential to the delimited area, over a defined range of potentials, andthe monitoring the rate of electron transfer (current) at eachpotential. The variation of electrical potential is analogous to thetaking of a series of optical measurement of a colored indicator atvarying the wavelengths. The electrical potential applied to theelectrode drives (forces) the targeted electroactive species to gain orlose electrons (reduction or oxidation, respectively) at a given ratethat is indicative of the concentration of such electroactive chemicalspecies. Accordingly, the resultant current not only reflects the rateat which the electrons move across the electron/solution interface, butalso (when compared to a standard), the concentration of the analyte inthe test sample. These potentiostatic techniques can, thus, measure anychemical species that is electroactive, (e.g. that can be made to reduceor oxidize within the environment of an electrochemical cell)

One of the preferred designs of the test strip useful in theelectrochemical quantitative analysis system of this invention hasmultiple components and multiple functional areas to (a) control thevolume and rate of absorption of sample by the test strip; (b)accommodate the controlled interaction of the sample and a labeledsubstance with an immobilized binding substance within a delimited areaof the test strip; (c) effect the separation of the labeled substancefrom the endogenous components of the sample; (d), the concentration ofthe labeled substance within a delimited area of the test strip forelectrochemical processing; and, (e) the quantitative determination ofthe analyte of interest by electrochemical means.

In another of the preferred embodiments of the foregoing test stripdesign, the sample and the labeled substance can be combined within abibulous pad (e.g., fiberglass) that is maintained in fluidcommunication with a solid phase affinity chromatographic test medium.Upon transfer of the sample thereto, the labeled substance isreconstituted, and the resultant labeled substance and/or a complexthereof which is formed with the analyte of interest, drawn into suchtest medium. As this fluid and its constituents, (e.g. sample, test kitreagent and reaction products thereof), diffuse into and within theaffinity chromatographic test medium, the labeled substance becomesbound to a delimited area (also hereinafter “test site”) along thisfluid pathway. Typically, this delimited area can be defined by means ofan immobilized binding substance that is specific for interaction withthe analyte, an analyte mimic or a complex of the analyte and/or alabeled ligand (e.g. by binding to an epitope on the analyte). As theanalyte, analyte mimic or a complex of the analyte and/or a labeledligand becomes increasingly concentrated at the test site, it can bemeasured and the amount thereof correlated with the analyte in the testsample.

The advantages of such controlled potential (potentiostatic) techniquesinclude high sensitivity, selectively toward an electroactive species, awide linear range, portable & low cost instrumentation and speciationcapability.

In order to accommodate multiple functional components within theintegral device of the preferred test strip design of this invention,all of the components are preferably arranged/mounted on a commonsupport or backing layer (typically an inert plastic, e.g. Mylar). Thesubstrate of this test device can be pre-printed with a conductivematerial to afford electrical contact (direct or inductive coupling)between the affinity chromatographic test medium of the test strip andan electrochemical analyzer that is configured to measure subtleelectrical changes within the delimited areas in the test strip. As morefully set forth in the description of the Figures, which follows, thissupport layer is typically imprinted at two or more locations with aconductive material or metal salt, to form what are referred tohereinafter as “electrodes”. These electrodes are spatially arrangedalong the support layer to coincide with delimited areas of the solidphase medium. The electrode coincident with the delimited area of thetest site is referred to as the “working electrode”. Depending upon theelectrochemical method of analysis, the test strip generally requires atleast one additional (e.g. “reference” electrode) to form the plates ofan electrochemical cell.

After having concentrated the labeled substance within the delimitedarea of the test site, it can be measured by a number of electrochemicaltechniques. As noted above, it may be desirable, preliminary toperforming such measurement, to first isolate the label from the complexby “pre-concentration” thereof on the working electrode. This“pre-concentration” process can involve the release/displacement of thelabel from the complex and the capture of the label in a reduced form(e.g. metal film (or metal surface-active complex)) on the workingelectrode where it is more electrochemically available or active. Uponcompletion of this pre-concentration process, the resultant metal film(or metal surface-active complex) can be subjected to potentiostaticelectrochemical quantitative analysis by anodic stripping voltammetry.In the context of this preferred analytical system (e.g. potentiostaticelectrochemical quantitative analysis by anodic stripping Voltammetry),the label is reoxidized, under electrochemical quantitative analyticalconditions, and this electrochemical transition of the label (from thereduced to the oxidized state) is monitored. The current signal that isgenerated during this transition (peak and area) is dependent upon anumber of system variables, the characteristics of the metal label andthe electrode geometry. In each instance, however, these variables canbe optimized by empirical adjustment; and, the analysis conditionstailored to correspond to standard curves which are used to correlateelectrical signal response to analyte concentration. More specifically,FIG. 4 is a graphical depiction of current signal response of a leadmetal film on a carbon electrode to anodic stripping analysis from lowto high concentration of lead. Similarly, FIG. 5 illustrates thevariation in signal (current) intensity, as a function of the durationof “pre-concentration” interval, (prior to electrochemical analysis) indetection of bismuth. In the graphical depiction of this variable shownin FIG. 5 the longer the pre-concentration interval (which varied from 1to 15 minutes) the greater the current signal intensity. Similarcorrelation/optimization in analytical process conditions is made foreach of the labels that are selected and standard curves for eachcreated, as appropriate, to correlate with the dynamic range ofconditions likely to be encountered for a given analyte and over a rangeof concentration. FIG. 6 shows the responses of multiple labels in adetecting window in one solution containing Indium, Lead, Copper andBismuth. Using multiple labels in this invention, analytes of interestin one sample solution can be simultaneously quantified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration, in perspective, of test strip component ofthe solid phase, electrochemical, quantitative analysis system of thisinvention (A: a two electrode system; B: a three electrode system).

FIG. 2 is a cross-section of the test strip of FIG. 1 along plane 22.

FIG. 3 is an illustration of the test device of this invention.

FIG. 4 graphically depicts a current signal response curve of abismuth/mercury amalgam on a carbon electrode to anodic strippinganalysis over a range of concentrations (1 ng/ml to 50 ng/ml ofpre-concentrated bismuth metal label).

FIG. 5 graphically depicts a current signal response curve illustratingthe variation in signal (current) intensity, as a function of theduration of “pre-concentration” interval, (prior to electrochemicalanalysis) in detection of bismuth. In the graphical depiction of thisvariable shown in FIG. 4, the longer the pre-concentration interval(which varied from 1 to 15 minutes) the greater the current signalintensity.

FIG. 6 graphically depicts a current signal response curve illustratingthe responses of multiple labels in a detecting window in one solutioncontaining Indium, Lead, Copper and Bismuth metal labels.

FIG. 7 graphically depicts a current signal response curve illustratingthe electrochemical analysis of standard Myoglobin antibody solutions atdifferent concentration in accordance with the system, method and teststrip (FIGS. 1 & 2) of this invention.

FIG. 8 graphically depicts a current signal response curve illustratingsix repeat measurements of a 2.5 ng/ml HBsAg in serum sample using thesystem, method and test strip (FIGS. 1 & 2) of this inventions.

FIG. 9 graphically depicts a current signal response curve illustratingthe measurement of HBsAg in a serum sample, using a HBsAg antibodylabeled by Lead, Copper and Bismuth ions.

FIG. 10 graphically depicts a current signal response curve illustratingmeasurement of HCG (left), HBsAg (right) and control label(middle) inserum sample, using the system, method and test strip (FIGS. 1 & 2) ofthis invention.

FIG. 11 graphically depicts a current signal response curve illustratingcompetitive detection of human NPOR (25 mer oligonucleotide), A (upper)is the signal of blank solution (without human NPOR) and B(lower) is thesignal of solution containing human NPOR.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

One of the distinct advantages of the system, method and test strip ofthis invention, is the unique ability to perform concurrentelectrochemical quantitative analysis for multiple analytes within agiven test sample. Thus, it is possible to perform a drug of abuse panelof assays on a given sample by simply utilizing a different labeledsubstance for each of the analytes of interest within a given sample.Accordingly, it is understood that the discussion which follows is notintended to either limit the scope or application of the invention toanalysis for a single analyte; and, that such description is onlylimited to analysis to a single analyte for ease of understanding andexemplification.

As noted above in summary fashion, and once again emphasized, theelectrochemical, quantitative analysis system of this invention is basedupon the initial concentration of a labeled substance within a delimitedarea of solid phase test environment; and, thereafter, causing anelectronic transition in the form of the label, so as to produce ameasurable electrical signal within the environment being monitored,which can be correlated with the quantity of the analyte within the testsample.

In certain instances, it may be appropriate (as an intermediate step inthe analysis routine) to release/displace the label from the labeledsubstance, and thereby convert it, or cause it to react with anothersubstance, that permits greater accessibility for electrochemicalquantitative analysis. Where for example, the labeled substance islabeled with a metal, (e.g. labeled ligand), the isolation andconcentration thereof within the delimited area of the test stripgenerally requires release and/or displacement thereof from its ligandconstituents and the conversion, or reaction thereof, so as tocause/effect its transition to a form (e.g. readily oxidizable orreducible state), which is more readily available for electrochemicalstate to another. In one of the preferred embodiments of this invention,the labeled ligand is comprised of a functional group that forms achelate-like coupling to the metal label. This coupling is, however, pHsensitive, and thus, the metal label can be released/displaced from thecomplex, after concentration within a delimited area of the solid phase,by simply contact thereof with an acidic salt solution.

Alternatively, where the labeled ligand is comprised of an indicator(e.g. metal) encapsulated within a liposome, such as described inCampbell, et al, U.S. Pat. No. 4,703,017 (previously discussed herein),that is in turn conjugated to a ligand, the integrity of liposomecapsule can be readily compromised/dissolved with a number of commonlipid solvents (e.g. detergents). The metal can, thus, be released fromthe ligand constituents of the labeled ligand, and made more readilyaccessible/available for quantitative analysis thereof.

Where the electrochemical quantitative analysis utilizes anodicstripping voltammetry, as the analytical method of choice, thereleased/displaced metal label can be reacted with mercuric ions withinthe acidic salt solution to form an amalgam on the working electrodecontiguous with the delimited area of the solid phase medium. Theelectrochemical reduction of the metal label is, thereafter, reversedunder subsequent electrochemical quantitative analysis, and the subtleelectrical changes monitored during such transition and correlated withthe presence and concentration of analyte in the test sample.

In order to provide for ease of use and portability of the system andmethods of this invention, the quantitative analysis system of thisinvention utilizes an affinity chromatographic test strip wherein adelimited area thereof is configured to concentrate the analyte ofinterest. The test devices illustrated in FIGS. 1 & 2 are representativeof a preferred embodiment of this test device.

The test device depicted in FIGS. 1 & 2 is initially composed of anaffinity-chromatographic test strip and an electrochemical detectorsystem.

The affinity-chromatographic test strip includes a plurality of discreteareas; and, is preferably, a composite of at least two (2) and morepreferably three (3) discrete components, in fluid communication withone another. As shown in FIG. 1, the test strip comprises a samplecollection pad (12), having labeled substance (14) (e.g. labeled ligandor labeled analyte mimic) pre-disposed within the sample collection pad,either coincident with or contiguous with the sample collection pad(14), a membrane (22), having substance (18) (e.g., ligand or analytemimic) pre-coated on the membrane, and a absorbent pad which is used toforce migrating of sample solution. In the preferred embodiments of thisinvention, the labeled substance is lyophilized (to permit stabilizationthereof prior to use); and, thereafter reconstituted upon contact withthe liquid sample.

Within the context of the system and analytical method of thisinvention, a “labeled substance” can include any suitable electroactivematerial that can be isolated within a delimited area of the solidphase, under assay conditions, or which can release an electroactivecomponent within such delimited area, (also collectively hereinafterreferred to as the “electroactive species”) and, thereafter, undergo anelectrochemical transition (e.g. redox) incidental to electrochemicalquantitative analysis. The observed measurement (transition) of theelectroactive species can be directly or inversely related, (e.g. bycomparison to a standard curve), to the concentration of the analyte ofinterest in the test sample.

In the preferred embodiments of this invention, the labeled test kitreagent is typically comprised of a protein or ligand associated with ametal label. Upon contact thereof with the sample, this metal labeledreagent interacts with the analyte of interest in the sample to form acomplex. A labeled substance, which can be utilized in the affinitychromatographic isolation/concentration of the analyte of interest,comprises and a protein/ligand and a metal label that is preferableprepared in accordance with the procedures described in U.S. Pat. No.4,732,974 and in the technical literature, Gary, et al, CovalentAttachment of Chelating Groups to Macromolecules, Biochem. And Biophys.Res. Comm., Vol. 77, No. 2(1977)pp581-585(which is herein incorporatedby reference in its entirety). In brief, a metal label can be conjugatedto the protein/ligand specific for the analyte, by initial modificationof the protein/ligand by covalently binding thereto of an “exogenouschelating group” having an affinity for the metal label. The chelatinggroups specific for the metal indicator of interest, can be covalentlycoupled directly to the binding protein/ligand or indirectly coupledthrough an intermediate or linking group. The coupling site must ofcourse be remote from the immuochemically active site that is specificfor the analyte of interest. Metal labels that are particularly suitablefor use in the synthesis of the labeled substances, that can be used asthe test kit reagents in the systems and methods of this invention,include Mg, Al, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y,Zr, Mo, Tc, Pd, Cd, In, Sn, Sb, Ba, Hf, W, Re, Hg, Tl, Pb, La, Ce, Rh,Pr, Nd, Sm, Eu, Gd, Th, Dy, Ho, Er, Yd, Th, U, Pu and the isotopesthereof.

Another of the labeled substances that can be utilized in the affinitychromatographic isolation/concentration of the analyte of interestcomprises an analyte mimic coupled to a liposome. The preparation ofliposome labeled immunoreagents, and their use in affinitychromatographic assays, is well-known and documented in the technicalliterature, Roberts, et al, Investigation of Liposome-BasedImmunomijuation Sensors for the Detection of Polychlorinated Biphenyl,Anal. Chem, Vol. 57, No. 5 (Feb. 1, 1995) pp. 482-491.

The labeled substance and/or complex of the labeled substance and theanalyte, is carried by diffusion/capillary action from the sampleapplication pad along the fluid pathway of the solid phase affinitychromatographic medium (16) to a downstream test site (18) where it isconcentrated and immobilized. This can be accomplished in thetraditional manner, by the use, for example, of a companion test kitreagent in the form of an immobilized binding protein or otherexpedient, that is specific for interaction with the analyte/metalcomplex. Thus, as the labeled substance and/or complex migrates/diffusesalong the fluid pathway of the test device, it encounters theimmobilized binding substance, and is thereupon captured so as toaccumulate at the test site. After a suitable reaction period,sufficient analyte/labeled substance has accumulated in the delimitedarea of the test strip to provide the requisite amount to be detectedand quantified by electrochemical analysis.

The electrochemical detector system (26) is prepared by printing twoelectrodes (for a two electrode system) or three electrodes (for a threeelectrode system) on a matrix material(e.g., MYLAR, plastic or othermaterial which is relatively electrically (non-conductive) insulating. Asupporter (24) with double side adhesive is used to combine theaffinity-chromatographic test strip and the electrochemical detectorsystem. A hole in the supporter is between the electrochemical detectorsystem and the test zone (18) in the membrane.

The detection and measurement of the quantity of the analyte in thesample is accomplished by means of equipment that can measure subtleelectrochemical changes associated with an electroactive species, as forexample, changes in current or voltage caused by the transition of suchspecies form the reduced to an oxidized form. In practice, this can beaccomplish by insertion of the affinity chromatographic test strip ofthis invention into a suitable measuring device and thereby electricallycoupling the device, to electrodes (26) at the test site (18). Theelectrical coupling of the monitoring instrument to the affinitychromatographic test strip can be by direct (physical contact) orindirect (inductive) coupling of the instrument to the electrodesassociated with the delimited area/test site of the test device. Thetest device is also electrically concurrently coupled to the test stripat another location (28), to provide a point of reference or internalstandard for comparison of readings at the test site. Thereafter, anelectrical potential is applied to the test site to excite/convert thelabel from one form/state to another, and such transition monitored.This transition is manifest in a number of ways, depending upon thenature and/or magnitude of the electrical force applied to the testsite, and other system variable relating to pre-concentration and theinherent characteristics of the label.

Another design for this test device is depicted in FIG. 3 wherein theaffinity-chromatographic test strip and electrochemical detector systemare placed within a housing which is composed of an isolator (30), a topsection or cover (32) and a bottom section or base (34); the base beingadapted for coupling to the cover, so as to seal and protect theaffinity-chromatographic test strip and electrochemical detector systemtherein and, thus, provide a tamper resistant environment. The isolator(30) is funnel shaped and adapted to direct test kit reagents onto thetest site and thereafter be inserted into the housing through a hole inthe cover of the housing, whereby it punches out a defined area withindetection zone (test site) of membrane so as to isolate a delimited area(volume) of membrane, in relation to the volume of the test kitreagents, for pre-concentration of the indicator.

EXAMPLES

The Examples that follow further define, describe and illustrate anumber of the preferred embodiments of the test kit reagents andelectrochemical methods of analysis of this invention. Apparatus andtechniques used in the synthesis of the materials described/used inthese Examples are standard, unless otherwise indicated. Parts andpercentages appearing in such Examples are by weight, unless otherwiseindicated.

Example 1

1. Preparation of ions labeled proteins (e.g., antibody and antigen)

Diethylenetriamine pentaacetic acid and Triethylamine were dissolved inwater with gentle heating. The resulting solution was lyophilized toyield a glassy residue and then such residue was dissolved inacetonitrile with gentle heating. This solution was added toisobutychloroformate in an ice bath during stirring for completereaction. The resulting anhydride Diethylenetriamine pentaacetic acidwas then reacted with protein in a protection solution at a certainmolar ratio at room temperature. The reaction mixture was then dialyzedagainst a citrate buffer. An excess of label solution was added to thedialyzed solution and incubated at room temperature. The labeled proteinwas dialyzed against phosphate buffer solution. The proteins (AntiHBsAgAb, AntiHCG Ab, AntiMyoglobin Ab, AntiTroponine I Ab, G-SAG, IgG-Fc, HIVand HCV etc.) are well labeled by metal ions (e.g., Bismuth, Lead,Indium, Thallium and copper) using the method above in our Lab. Thelabeled protein was used to prepare a solid phase & affinitychromatographic test strip.

2. Preparation of affinity chromatography solid phase test device

Two conductive deposits or areas (for a two electrode system),corresponding to the test site (carbon ink electrode), and the referencesite (Ag/AgCl ink electrode) are ink jet printed on a polyester lamina.The size of each printed area is 4×4 mm and approximately 5/1000 inchthick.

A membrane, having an immobilized ligand predisposed at the test site(detection zone), was laminated to the backing layer so as to align thetest site over the carbon ink electrode system. A fiberglass pad(containing the label/ligand conjugate) and excess fluid absorbentmedium are each positioned relative to each end of the laminate to formthe assembled test strip. The test device fabricated in accordance withthis Example is depicted in FIGS. 1, 2 & 3.

3. Measurement of the analyte

In the course of performance of an assay for HCG, HBsAg, Myoglobin ormultiple analytes utilizing the foregoing metal ion labeled ligand(s)and affinity chromatographic solid phase test device, a liquid sample isfirst applied to the sample application region followed by the migrationof the liquid sample by capillary action along the affinitychromatographic strip towards the other end of the affinitychromatographic strip. As the liquid migrates within the sampleapplication pad, and reconstitutes the labeled ligand(s) containedtherein, analyte(s) in the sample react with the labeled ligand(s)forming a complex(es) which is transferred to and drawn into theaffinity chromatographic test medium. When the complex reaches thedetection region (test site), the analytes in the complex react with theimmobilized ligand(s) in the detection region forming a labeledligand-analyte-ligand complex(es), while the unbound constituents andfluid fraction continue to be drawn and flow into the absorbent mediumat the opposite end of the device. The formation of the complex in thedetection region is then registered and the labeler metal ionsquantified by the Square Wave Stripping Voltammetry (SWSV). As describedabove, this stripping procedures involves preconcentration (or formationadsorptive accumulation of a surface-active complex) and determinationof the metal ions.

The foregoing analysis is particularly advantageous where the testsample is a turbid solution that can interfere with more traditionaloptical measurement techniques, such as a blood or a urea samplesolution FIG. 7 graphically illustrates an electrochemical analysis of astandard Myoglobin antibody solution in different concentration inaccordance with the system, method and test device (FIG. 3) of thisinvention. The low detection limits and sensitivity of this assay due,in part, to the high molar labeling ratio of lead ions relative to theanti-Myoblobin antibody, and the duration of the interval ofpre-concentration of the lead on working electrode. FIG. 8 shows repeatmeasurements of 2.5 ng/ml HBsAg in serum sample in accordance with thesystem, method and test device (FIG. 3) of this invention. Areproducible response is observed over six repeat measurements. Also,simultaneous detection for multiple analytes of interest is availablewith the system, method and test device (FIG. 3) of this invention. FIG.9 is the response of HBsAg in serum sample, using a HBsAg antibodylabeled by Lead, Copper and Bismuth ions, different ion can be seen inthe response curve, which shows that complexes(Anti-HBsAg antibody-HBsAgantigen-Anti-HBsAg antibody-Anti-HBsAg antibody labeled different metalion complexes) were formed and detected in the test zone. FIG. 10 is asimultaneous detection for HCG(left) and HBsAg(right) in serum sample,using the system, method and test device (FIG. 3) of this invention, thepeak at middle is a control test which tells that the device is work.

Example 2 Nucleic Acids (Oligonucleatides, DNA and RNA) Sensor

1. Preparation of ions labeled oligonucleatide

Diethylenetriamine pentaacetic acid and Triethylamine were dissolved inwater with gentle heating. The resulting solution was lyophilized toyield a glassy residue and then such residue was dissolved inacetonitrile with gentle heating. This solution was added toisobutychloroformate in an ice bath during stirring for completereaction. The resulting anhydride Diethylenetriamine pentaacetic acidwas then reacted with oligonuleatides at a certain molar ratio at roomtemperature. The reaction mixture was then dialyzed against a trisbuffer. A excess of label solution was added to the dialyzed solutionand incubated at room temperature. The labeled oligonucleotide wasdialyzed against tris buffer solution. The labeled oligonucleotide wasused to prepare a solid phase & affinity test sensor.

2. Preparation of affinity chromatography solid phase test device

Two conductive deposits or areas (for a two electrode system),corresponding to the test site (carbon ink electrode), and the referencesite (Ag/AgCl ink electrode) are ink jet printed on a polyester lamina.The size of each printed area is ˜4×4 mm and approximately 5/1000 inchthick.

A membrane, having an immobilized ligand predisposed at the test site(detection zone), was laminated to the backing layer so as to align thetest site over the carbon ink electrode system. A fiberglass pad(containing the label/oligonucleatide conjugate) and excess fluidabsorbent medium are each position relative to each end of the laminateto form the assembled test strip. The test device fabricated inaccordance with this Example is depicted in FIGS. 1, 2 & 3.

3. Measurement of the human NPOR

In the course of performance of an assay for human NPOR utilizing theforegoing metal ion labeled oligonucleatide and affinity chromatographicsolid phase test device, a liquid sample contain the oligonucleatide ofinterest is first applied to the sample application region followed bythe migration of the liquid sample by capillary action along theaffinity chromatographic strip towards the other end of the affinitychromatographic strip. As the liquid migrates within the sampleapplication pad, the labeled oligonucleatide imbedded in the contact padis carried out and interacts competitively with the complementoligonucleatide coated at the test zone to the oligonucleatide ofinterest in sample solution, forming a labeledoligonucleatide-complement oligonucleatide complex andoligonucleatide-complement oligonucleatide complex, while the unboundconstituents and fluid fraction continue to be drawn and flow into theabsorbent medium at the opposite end of the device. The formation of thecomplexes in the detection region is then registered and the labelermetal ions quantified by the Square Wave Stripping Voltammetry (SWSV).As described above, this stripping procedures involves preconcentration(or formation adsorptive accumulation of a surface-active complex) anddetermination of the metal ions. FIG. 11 shows competitive detection ofhuman NPOR (25 mer oligonucleotide), using the system, method and testdevice (FIG. 3) of this invention. A(upper) is the signal of blanksolution (without human NPOR) and B (lower) is the signal of solutioncontaining human NPOR. Such result offers a method to detect andsequence DNA.

What is claimed is:
 1. A system suitable for concurrent electrochemical,quantitative determination of multiple analytes in a common test fluidsample, comprising: A. at least one test kit reagent comprising alabeled substance that is both specific for interaction with an analyteof interest or mimicking of an analyte of interest within said system,and electrochemically active so as to be capable of electrolyticreduction and oxidation, under analysis conditions; B. a solid phase,porous, electrochromatographic medium having a common, linear fluidpathway defined therein, said linear pathway having a delimited areacontaining at least one immobilized binding material specific forbinding to at least one labeled substance or to a complex of a labeledsubstance and an analyte suspected of being present in said common fluidtest sample; C. means for combining said test kit reagent(s) and acommon fluid test sample suspected of containing one or more analytes ofinterest within said solid phase, porous electrochromatographic medium,so as to cause said common fluid sample containing said labeledsubstance(s) and/or a complex of a labeled substance(s) and one or moreof said analytes of interest, to flow along said fluid pathway of saidmedium to said delimited area of said medium and thereby contact saidimmobilized binding material within said delimited area of said medium,under binding conditions, whereby said labeled substance(s), and/or acomplex of a labeled substance(s) and an analyte of interest, is boundby said immobilized binding material within said delimited area of saidmedium; D. electrode means coincident with and in spaced-apartrelationship with said delimited area of said medium, said electrodemeans including at least one working electrode and second electrodeselected from the group consisting of a reference electrode and acounter electrode; E. means for displacement, under electrolyticconditions, of said label(s) from each of said labeled substance(s),and/or from each of said complex of a labeled substance and analyte(s),bound within said delimited area of said medium, so as to cause saidlabel to form a deposit upon said working electrode of said electrodemeans; F. means for activation of said working electrode, understripping conditions, so as to effect oxidation of said deposit andthereby produce a current signal response indicative of theconcentration of each said analyte(s) of said common test fluid and G.means for correlation of said current signal response with a standard orreference.
 2. The system of claim 1, wherein the solid phase, porouselectrochromatographic medium includes a second delimited area and asecond means for establishing electrical contact therewith under assayconditions, and means for comparison or reference thereof to saidelectrochemical changes, characteristic of each label.
 3. The system ofclaim 1, wherein the labeled substance comprises a metal selected fromthe group consisting of Mg, Al, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu,Zn, Ga, Sr, Y, Zr, Mo, Tc, Pd, Cd, In, Sn, Sb, Ba, Hf, W, Re, Hg, Tl,Pb, La, Ce, Rh, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yd, Th, U, Pu andthe isotopes thereof.
 4. The system of claim 1, wherein the labeledsubstance comprises an analyte mimic, a complex of an analyte mimic witha labeled ligand or a liposome, wherein said substance iselectrochemically active under electrochemical analytical conditions. 5.The system of claim 1, wherein the electrochemical analytical conditionscomprise potentiostatic measurement of an electrochemically activesubstance indicative of the analyte of interest.
 6. The system of claim1, wherein said means for combining said test kit reagent(s) and acommon fluid test sample suspected of containing one or more analytes ofinterest within said solid phase includes (a) a housing having a topsection and base section, wherein at least one of said top section orbase section has means for coupling said base to said top section, so asto seal said solid phase with said housing, and thereby align said solidphase relative to a series of apertures in said top section of saidhousing, and (b) means, associated with an aperture of said housing, forisolating a test site within said solid phase, so as to direct test kitreagents onto said test site and thereby confine interaction of test kitreagents and said fluid sample to within a delimited area of said solidphase.